The translation machinery is able to catalyze the mRNA-template directed synthesis of polypeptides with an error rate of only one in every 1,000 to 10,000 peptide bonds synthesized. Significant advances in the last decade not only in the determination of high resolution structures of the bacterial translation machinery but also the precise mechanism of the elongation cycle of translation make it possible for the first time to examine how the translation machinery is able to work with the chemically diverse 20 natural amino acids, but at the same time have such a low error rate. Understanding the substrate selectivity of the translation machinery is of major importance both because translation is essential to cellular function and because it speaks to the question of how complex biomolecular machines carry out sophisticated functions. Despite the amount of data provided by the kinetic model of the elongation cycle, several lines of evidence suggest that it cannot fully account for substrate selection. While it is apparent that the translation machinery is sensitive to more than just the codon anticodon interaction, there has not been a decisive set of experiments to demonstrate this sensitivity from the perspective of selection, nor is there a model that would allow us to predict how these various other factors would affect translation. We currently find ourselves in a unique position to fill this void. AIM 1: We propose to test the hypothesis that the aminoacy-tRNA combinations are discriminated during the elongation cycle using peptide incorporation yield and competition assays we've developed in conjunction with single molecule FRET experiments. AIM 2: We propose to test the hypothesis that discrimination of aminoacyl-tRNA combinations occurs after peptide bond formation. We also propose to test which aspects of the aminoacyl tRNA combinations are important for discrimination. There are some early findings in the literature indicating that malfunctions at the level of translation are involved disease pathology;for example, mutations in the glycine synthetase are linked to Charcot-Marie- Tooth disease. Further elucidation of the selection ability of the translation machinery will help us understand how these malfunctions are handled by the translation machinery, and will perhaps reveal other important links to pathology.